RNA virus populations are complex distributions of closely related but nonidentical
variant genomes termed quasispecies. The large heterogeneity in quasispecies
is due to the low fidelity of viral RNA polymerases and the lack of error-repair activities
during replication. Studies on genome replication with high error rates predict the
existence of an error threshold above which the maintenance of the genetic information
would not be possible. The quasispecies structure would be lost when the error rate of
replication crosses the error threshold, in a process that has been termed entry into error
catastrophe. This fact opens interesting possibilities for the design of a new antiviral
strategy based on inducing viral error catastrophe by forcing viruses to replicate in the
presence of mutagens. The term lethal mutagenesis is referred to as viral extinction
through error catastrophe. A critical question for the application of error catastrophe is
whether mutations can occur that render viruses resistant to mutagenic agents. The main
objective of this Ph. D. Thesis is to understand the mechanisms underlying resistance of
RNA viruses to mutagenic agents and the evolutionary implications of quasispecies
dynamics.
The nucleoside analogue ribavirin (R) is mutagenic for foot-and-mouth disease
virus (FMDV). We have selected FMDV with amino acid substitution M296I in the
viral polymerase (3D) by passaging FMDV in the presence of increasing concentrations
of R. Measurements of progeny production and viral fitness with chimeric viruses in the
presence and absence of R documented that 3D substitution M296I conferred FMDV a
selective replicative advantage in the presence of R but not in the absence of R. Purified
mutant polymerase with I296 showed a decreased capacity to use ribavirin triphosphate
(RTP) as substrate as compared with the wild type enzyme. The results suggest that
M296I has been selected because it attenuates the mutagenic activity of R on FMDV.
Replacement M296I is located within a highly conserved stretch in picornaviral
polymerases, which includes residues that interact with template-primer and with the
incoming nucleotide, according to the three-dimensional structure of FMDV 3D. Given
that a 3D substitution, distant from M296I, was associated with resistance to R in
poliovirus, the results indicate that picornaviral polymerases include different sites that
can alter the interaction of the enzyme with mutagenic nucleoside analogues.
Implications for lethal mutagenesis are discussed.